JP2011115120A - Method for preparing microorganism-carrying carrier for forming methane gas from lignocellulose-based biomass - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prepare a microorganism-carrying carrier promoting the methane fermentation of a lignocellulose-based biomass, while suppressing energy consumption in a preceding stage for providing the lignocellulose-based biomass to the methane fermentation. <P>SOLUTION: The method for preparing the microorganism-carrying carrier includes a process of adding a carrier carrying the microorganisms into a suspension added with a bacterial population containing at least anaerobic cellulose-decomposing bacteria and acetic acid-assimilating methane bacteria, and also progressing the decomposition reaction of the lignocellulose-based biomass with the microorganism population by adding the lignocellulose-based biomass into the suspension. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing a microorganism-supporting carrier for producing methane gas from lignocellulosic biomass. More specifically, the present invention relates to a method for producing a microorganism-supporting carrier suitable for producing methane gas from lignocellulosic biomass, particularly rice straw, and a method for producing methane gas using the microorganism-supporting carrier.

  Lignocellulosic biomass, which is a biomass containing cellulose, hemicellulose, and lignin, is a major biomass present on the earth, and its expansion as a carbon neutral energy source is desired.

  Examples of lignocellulosic biomass include softwood materials, hardwood materials, non-tree materials, and wastes thereof. In particular, rice, barley, wheat, corn, and other straws, which are non-wood wastes, are produced in large quantities every year in the world and are difficult to dispose of. This is no exception in Japan. In Japan, a large amount of rice straw is generated every year as a non-edible part of rice, the main agricultural product, and it is difficult to dispose of it. Therefore, there is a strong demand for effective use of rice straw.

  By the way, as one method of using lignocellulosic biomass as an energy source, it has been proposed to generate methane gas using lignocellulosic biomass as a carbon source by methane fermentation and to use this methane gas as energy. However, since the biodegradation of cellulose and hemicellulose is inhibited by lignin, which is a hardly decomposable substance contained in lignocellulosic biomass, methane gas cannot be produced with high efficiency even when lignocellulosic biomass is directly subjected to methane fermentation. That was a problem.

  Therefore, as a method for solving this problem, a method of subjecting lignocellulosic biomass to some kind of pretreatment to make it readily biodegradable and then subjecting it to methane fermentation has been proposed.

  For example, in Patent Document 1, lignocellulosic biomass is rotted using white rot fungi, then the rotified biomass is placed in a pressure resistant vessel, and high-temperature (180 ° C or lower) steam is introduced and held for a certain period of time. After that, the pressure vessel is released to the atmosphere at once, and the biomass is crushed (steamed and crushed), and the crushed biomass is subjected to methane fermentation.

  Further, in Patent Document 2, by means of an ozone reactor having means for drying / miniaturizing lignocellulosic biomass and ozone reaction means for decomposing the contained lignin by reacting the biomass in the drying / miniaturization process with supply ozone, Lignocellulosic biomass is pretreated and then subjected to methane fermentation.

JP 2008-237164 A JP 2005-81317 A

  However, in the method described in Patent Document 1, energy for maintaining an environment (temperature / humidity) suitable for white rot fungi when performing the rot treatment with white rot fungi, and high-temperature steam used for steaming explosion treatment are used. Since it requires energy to generate it, it had a problem that energy was consumed before lignocellulosic biomass was subjected to methane fermentation and energy production efficiency from lignocellulosic biomass was reduced. .

  Also in the method described in Patent Document 2, energy for generating ozone is required, and energy is consumed in the previous stage of subjecting lignocellulosic biomass to methane fermentation, and energy production efficiency from lignocellulosic biomass. Has the same problem as Patent Document 1 in that it decreases.

  In addition, as described above, lignocellulosic biomass is easy to obtain because it is the main biomass present on the earth, and especially for rice straw and other straw, there is a demand for its effective use. . Under such circumstances, if energy can be efficiently generated from lignocellulosic biomass while suppressing energy consumption in the previous stage of lignocellulosic biomass for methane fermentation, new energy can be established. This is extremely effective as a production technology.

  Therefore, the present invention provides a method for producing a microorganism-supporting carrier capable of promoting methane fermentation of lignocellulosic biomass while suppressing energy consumption in the previous stage of subjecting lignocellulosic biomass to methane fermentation. With the goal.

  In addition, the present invention provides a method capable of efficiently generating methane gas by promoting methane fermentation of lignocellulosic biomass while suppressing energy consumption in the previous stage of subjecting lignocellulosic biomass to methane fermentation. The purpose is to do.

  In order to solve such a problem, the inventors of the present application conducted intensive research. First, rice straw was added to the methane fermentation broth to perform methane fermentation. As a result, the hydraulic residence time was set to 15 days (organic load: 620 mg COD / L / day) and after 15 days had elapsed, the hydraulic residence time was set to 10 days (organic load: 970 mg COD / L / day). ), It was confirmed that the methane gas production rate gradually decreased. However, a similar experiment was conducted by adding a carbon fiber nonwoven fabric together with rice straw to the methane fermentation broth. Surprisingly, even if the hydraulic retention time was set to 10 days, the methane gas production rate did not decrease, and the experiment was conducted. It was confirmed that it continued to increase until the 45th day at the end.

  The inventors of the present application conducted various analyzes to confirm the difference in the methane fermentation state depending on the presence or absence of the addition of the carbon fiber nonwoven fabric. When the carbon fiber nonwoven fabric was not added, the inventors volatilized in the methane fermentation liquid. A large amount of sex fatty acid was accumulated. On the other hand, when a carbon fiber nonwoven fabric was added, almost no accumulation of volatile fatty acids was observed in the methane fermentation broth. From this, when lignocellulosic biomass is directly subjected to methane fermentation, it is easy to accumulate volatile fatty acids. It came to know that there is an effect which prevents accumulation | storage of a volatile fatty acid by adding to a liquid.

  Therefore, the present inventors have genetically engineered the bacterial flora adhering to the carbon fiber nonwoven fabric in order to find out why the volatile fatty acid accumulation can be prevented by adding the carbon fiber nonwoven fabric to the methane fermentation broth. Analyzed. As a result, it was confirmed that an acetic acid-assimilating methane bacterium capable of converting volatile fatty acid into methane gas is preferentially supported on the carbon fiber nonwoven fabric. Furthermore, it was also confirmed that the anaerobic cellulose-degrading bacteria are supported on the carbon fiber nonwoven fabric.

  Based on the analysis results, the inventors of the present invention supported an anaerobic cellulose-degrading bacterium on a hydrophobic carrier capable of supporting microorganisms such as carbon fiber nonwoven fabric, and also supported an acetic acid-assimilating methane bacterium to produce cellulose resolving power and cellulose. It was found that a microorganism-supporting carrier having the ability to suppress the accumulation of volatile fatty acids, which are degradation products of methane, and the ability to generate methane gas from volatile fatty acids can be produced. Furthermore, it has been found that methane gas can be efficiently generated from lignocellulosic biomass by performing methane fermentation of lignocellulosic biomass using this microorganism-supported carrier, and the present invention has been completed.

  That is, in the method for producing a microorganism-supporting carrier for producing methane gas from lignocellulosic biomass of the present invention, microorganisms are added to a suspension to which a microorganism community containing at least anaerobic cellulose-degrading bacteria and acetic acid-assimilating methane bacteria is added. In addition to adding a carrier that can be supported, a step of adding a lignocellulosic biomass to the suspension and advancing the decomposition reaction of the lignocellulosic biomass by a microbial community is included.

  Here, in the method for producing a microorganism-supporting carrier of the present invention, the microorganism community includes an anaerobic cellulose-decomposing bacterium and an acetic acid-assimilating methane bacterium, and at least one of a hydrogen-assimilating methane bacterium and a saccharolytic bacterium. It is preferable to contain.

  In the method for producing a microorganism-supporting carrier of the present invention, the suspension is preferably methane fermentation sludge or a methane fermentation sludge-containing liquid.

  Furthermore, in the method for producing a microorganism-supporting carrier of the present invention, the hydrophobic carrier is preferably a carbon fiber.

  In the method for producing a microorganism-supporting carrier of the present invention, the lignocellulose-based biomass is preferably straw and more preferably rice straw.

  Next, in the method for producing methane gas from lignocellulosic biomass of the present invention, lignocellulosic biomass is subjected to methane fermentation using the microorganism-supported carrier obtained by the production method of the present invention.

  In the methane gas production method of the present invention, a methane fermentation treatment is performed by introducing a hydrophobic carrier carrying acetic acid-assimilating methane bacteria and lignocellulosic biomass into a methane fermentation liquid.

  Here, in the method for producing methane gas of the present invention, the lignocellulosic biomass is preferably straw and more preferably rice straw.

  According to the method for producing a microorganism-supporting carrier of the present invention, a microorganism-supporting carrier capable of efficiently decomposing the lignocellulosic biomass for methane fermentation and generating methane gas can be produced. Therefore, it is possible to efficiently produce methane gas from lignocellulosic biomass without consuming energy at the stage before subjecting the lignocellulosic biomass to methane fermentation. Moreover, according to the method for producing a microorganism-supporting carrier of the present invention, there is an advantage that methane gas can be generated while decomposing lignocellulosic biomass in the process of supporting microorganisms on the carrier.

  Further, according to the methane gas production method of the present invention, since lignocellulosic biomass is directly subjected to methane fermentation, lignocellulosic biomass is consumed without consuming energy in the previous stage of subjecting lignocellulosic biomass to methane fermentation. It is possible to efficiently generate methane gas from biomass.

  Furthermore, according to the method for producing a microorganism-supporting carrier and the method for producing methane gas of the present invention, since the lignocellulosic biomass, which is the main biomass existing on the earth, is used as a raw material, The convenience of generation can be further increased. It is also possible to effectively use rice straw and other straw that is generated in large quantities every year in Japan and other countries around the world and whose disposal is a problem as an energy source.

It is a figure which shows the operating condition (OLR, HRT) of a reactor. It is a figure which shows the time-dependent change of a gas production | generation speed | rate. It is a figure which shows a time-dependent change of a volatile fatty acid (VFA) density | concentration. It is a figure which shows the difference in the COD removal efficiency in Example 1 and Comparative Example 1. It is a figure which shows the difference in SS removal efficiency in Example 1 and Comparative Example 1. It is a figure which shows the time-dependent change of the quantity of soluble sugar. It is a figure which shows the result of having analyzed the archaeal community structure by T-RFLP. It is a figure which shows the result of having analyzed the bacterial community structure by T-RFLP.

  Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings.

<Method for producing microorganism-supporting carrier>
The method for producing a microorganism-supporting carrier for producing methane gas from lignocellulosic biomass according to the present invention comprises supporting a microorganism in a suspension to which a microorganism community including at least an anaerobic cellulose-degrading bacterium and an acetic acid-assimilating methane bacterium is added. In addition to adding the carrier to be obtained, the method includes a step of adding lignocellulosic biomass to the suspension and advancing the decomposition reaction of lignocellulosic biomass by microbial community.

  The microbial community used in the present invention may include at least an anaerobic cellulose-degrading bacterium and an acetic acid-assimilating methane bacterium, or a microbial community consisting only of an anaerobic cellulose-degrading bacterium and an acetic acid-assimilating methane bacterium may be used. Alternatively, an anaerobic cellulose-degrading bacterium, an acetic acid-assimilating methane bacterium, and a microbial community including other microorganisms may be used. For example, the microbial community may be constituted only by artificially cultured anaerobic cellulose-degrading bacteria and acetic acid-assimilating methane bacteria, and includes both anaerobic cellulose-degrading bacteria and acetic acid-assimilating methane bacteria Sludge and soil may be used as a microbial community, or a microbial community formed by mixing sludge containing an anaerobic cellulose-degrading bacterium and soil and sludge or soil containing an acetic acid-assimilating methane bacterium may be used.

  As the anaerobic cellulose-degrading bacterium, known or novel microorganisms that exhibit cellulose resolving power in a methane fermentation process that proceeds in an anaerobic environment can be used. For example, microorganisms belonging to the genus Clostridium can be used, but are not limited thereto.

  By supporting an anaerobic cellulose-degrading bacterium on a carrier, cellulose resolving power can be imparted to the microorganism-supporting carrier.

  As the acetic acid-assimilating methane bacterium, a known or novel microorganism that exhibits the ability to generate methane gas from acetic acid in a methane fermentation process that proceeds in an anaerobic environment can be used. For example, a microorganism belonging to the genus Methanosarcina can be used, and Methanosarcina thermophila can be preferably used, but the acetic acid-assimilating methane bacterium is not limited thereto.

  By supporting acetic acid-assimilating methane bacteria on a carrier, methane gas can be generated from volatile fatty acids such as acetic acid, propionic acid and butyric acid, which are degradation products of cellulose, and volatile fatty acids are consumed and accumulated. Can be prevented. Furthermore, as a result of the consumption of volatile fatty acids, which are degradation products of cellulose, the reaction of generating volatile fatty acids from cellulose is likely to proceed. Therefore, the progress of the decomposition reaction of cellulose can be promoted.

  Here, the microbial community used in the present invention may further include at least one of hydrogen-utilizing methane bacteria and saccharolytic bacteria.

  As the hydrogen-utilizing methane bacterium, a known or novel microorganism capable of exhibiting the ability to generate methane gas from hydrogen and carbon dioxide in the methane fermentation process that proceeds in an anaerobic environment can be used. For example, microorganisms belonging to the genus Methanobacterium and microorganisms belonging to the genus Methanothermobacter can be used, and Methanobacterium formicicum and Methanothermobacter thermautotrophicus can be preferably used. However, the hydrogen-assimilating methane bacteria are not limited to these.

  By supporting the hydrogen-assimilating methane bacterium on a carrier, methane gas can be generated from hydrogen and carbon dioxide, which are decomposition products of cellulose. Further, as a result of consumption of hydrogen and carbon dioxide, which are decomposition products of cellulose, a reaction in which hydrogen and carbon dioxide are generated from cellulose easily proceeds. Therefore, the progress of the decomposition reaction of cellulose can be promoted.

  As the saccharolytic bacteria, known or novel microorganisms capable of exhibiting the ability to degrade saccharides such as glucose and the like, which are degradation products of cellulose and hemicellulose, in the methane fermentation process proceeding in an anaerobic environment can be used. . For example, a microorganism belonging to the genus Anaerobaculum, which is a glucose-degrading bacterium, can be used. However, saccharolytic bacteria are not limited to this.

  By supporting a saccharide-decomposing bacterium on a carrier, a reaction in which a saccharide such as glucose, which is a decomposition product of cellulose, is decomposed and consumed, facilitates a reaction in which saccharide is generated from cellulose. Therefore, the progress of the decomposition reaction of cellulose can be promoted.

  Here, methane fermentation sludge is suitable as the microbial community used in the present invention. Methane fermentation sludge usually contains anaerobic cellulose-degrading bacteria, acetic acid-assimilating methane bacteria, hydrogen-assimilating methane bacteria, and saccharolytic bacteria, and these carriers are supported on lignocellulosic biomass. It is possible to produce a microorganism-supporting carrier that can carry out methane gas production from an extremely efficient manner.

  In addition, when methane fermentation sludge is used as the microbial community, it has been confirmed that the bacterial community carried on the carrier is diversified. Therefore, it is conceivable that diversification of the bacterial community supported on the carrier contributes in some way to the improvement of methane gas production efficiency from lignocellulosic biomass.

  The suspension to which the microbial community has been added is prepared by adding the microbial community to, for example, water in which the microbial community has been dissolved, preferably water in which inorganic salts that are preferable for the growth of microorganisms are dissolved. The amount of the microbial community added is not particularly limited, but should be an amount that can increase the amount of microorganisms in the suspension as much as possible while ensuring sufficient fluidity of the suspension. Is preferred. For example, when methane fermentation sludge is used as the microbial community, a methane fermentation sludge-containing liquid with a microbial community: water = 4: 1 (volume ratio) is preferable, but the mixture ratio is limited to this. It is not a thing. Further, the methane fermentation sludge itself may be used as a suspension without adding water, or the methane fermentation sludge directly added with inorganic salts or the like preferable for microbial growth may be used as a suspension. May be.

  The lignocellulosic biomass used in the present invention is not particularly limited, and softwood materials, hardwood materials, non-tree materials, and wastes thereof can be used. Specifically, Japanese cedar, Scots pine, Japanese larch, Japanese black pine, Todomatsu, Japanese pine, Yew, Nezuko, Hari fir, Iramomi, Inu Maki, Fir, Sawara, Togasawara, Asunaro, Hiba, Tsuga, Kototsuga, Hinoki, Ichii, Inugaya, Spruce, Yellow Cedar (Beihiba), Lawson Hinoki (Beihi), Douglas Fir (Bay Pine), Sitka Spruce (Beisuhi), Radiata Pine, Eastern Spruce, Eastern White Pine, Western Larch, Western Fur, Western Hemlock and Tamarack, Asben, American black cherry, yellow poplar, walnut, cabbage cherry, zelkova, sycamore, silver cherry, tamo, teak, chinese elm, chinese maple, oak, hard maple Hickory, pecan, white ash, white oak, white birch, red oak, acacia and eucalyptus hardwood, rice, sugarcane, wheat, corn, pineapple, oil palm, kenaf, cotton, alfalfa, timothy, bamboo, sasa, sugar beet Non-tree materials such as these, and wastes thereof. Among them, rice, barley (barley, wheat), corn, etc., which correspond to non-wood waste, and are produced in large quantities every year in the world, can be used suitably. It is more preferable to use the generated rice straw.

  The hydrophobic carrier capable of supporting the microorganism used in the present invention has a three-dimensional structure capable of supporting the microorganism, for example, a carrier having a large surface area secured by a high porosity. it can. Specifically, for example, a porosity of 25% to 98% may be used, but a porosity of 50% to 98% can be used more preferably, and a porosity of 98% is more preferable. Can be used. And as a support | carrier raw material for ensuring hydrophobicity, the thing made from carbon, the product made from polyethylene, and a polypropylene can be used conveniently, The thing made from carbon can be used especially suitably. A carbon material is easy to secure a high porosity, and, for example, a carbon fiber nonwoven fabric is suitable because it can easily ensure a high porosity (98%) and can be obtained at low cost.

  The shape of the hydrophobic carrier capable of supporting the microorganism is not particularly limited, and may be, for example, a sheet shape, a spherical shape, a tube shape, or the like. In addition, the hydrophobic carrier capable of supporting microorganisms does not need to be entirely formed of the above material, and for example, a substrate whose surface is covered with the above material is used as a hydrophobic carrier capable of supporting microorganisms. Also good.

  The amount of lignocellulosic biomass added to the suspension to which the microbial community has been added varies optimally depending on the amount of microbial communities (number of microorganisms) contained in the suspension. Although it cannot be defined, the amount may be an amount that causes an organic load that does not decrease or stop the progress of the methane fermentation reaction by the microbial community in the suspension. The lignocellulosic biomass may be added as it is, but is preferably added after increasing the contact area with the suspension by pulverization to promote the decomposition reaction of cellulose. .

  The amount of the hydrophobic carrier that can support microorganisms to the suspension to which the microbial community has been added is not particularly limited, but if too much hydrophobic carrier that can support microorganisms is added to the suspension, Since it becomes difficult to ensure the fluidity of the suspension, it is preferable to add the suspension in a range that can ensure the fluidity of the suspension.

  By adding lignocellulosic biomass to the suspension to which the microbial community has been added, the decomposition reaction of lignocellulosic biomass by the microbial community in the suspension proceeds. As the decomposition reaction proceeds, an anaerobic cellulose-degrading bacterium and an acetic acid-assimilating methane bacterium, and further a hydrogen-assimilating methane bacterium and a saccharifying bacterium are gradually added to the hydrophobic carrier capable of supporting the microorganism. In particular, acetic acid-assimilating methane bacteria are preferentially supported. In addition, when methane fermentation sludge is used as the microbial community, various bacterial communities are supported on the carrier. In this process, methane gas can be generated while lignocellulosic biomass is decomposed. In addition, in order to advance the decomposition reaction of lignocellulosic biomass, the suspension is kept in an anaerobic state. Further, it is preferable to stir the suspension during the progress of the reaction.

  Here, the contact time between the suspension to which the microbial community is added and the hydrophobic carrier capable of supporting the microorganism is determined based on the amount of the microbial community in the suspension (number of microorganisms) and the added amount of lignocellulosic biomass (organic matter). Since the optimal time varies depending on the amount of load), it cannot be specified unconditionally. However, it is assumed that the total amount of lignocellulosic biomass as a substrate is converted to methane. The time until the generation is achieved, specifically, 70% or more of the gas generation amount (mL) calculated by multiplying the COD (mg conversion) applied by 0.35 times is achieved. It is preferable to set the time until the gas generation amount (mL) at least 0.245 times the COD (mg conversion) is achieved.

  According to the experiments by the inventors of the present application, for 250 mL of suspension added with 200 g of methane fermentation sludge, when the hydraulic residence time is 15 days and the organic load is 620 mg COD / L / day, the conversion is milligrams in 6 days. It has been confirmed that 70% or more of gas generation amount (mL) 0.35 times the COD of COD is achieved.

  Here, the organic load amount of the suspension may be always constant, but as time passes, the desired microorganisms are gradually supported on the hydrophobic carrier capable of supporting the microorganisms, and the methane fermentation treatment capacity is slightly reduced. Since it improves step by step, the organic load may be increased little by little. In this case, the contact time between the suspension to which the microbial community is added and the hydrophobic carrier capable of supporting the microorganism can be shortened.

<Methane gas production method>
By subjecting lignocellulosic biomass to methane fermentation using the microorganism-supported carrier obtained by the production method of the present invention, methane gas can be efficiently generated from lignocellulosic biomass.

  When lignocellulosic biomass is directly subjected to methane fermentation, volatile fatty acids accumulate in the methane fermentation liquid and the progress of methane fermentation is likely to be slowed or stopped. This has been a factor that makes it impossible to efficiently generate methane gas from lignocellulosic biomass. The microorganism-supporting carrier obtained by the production method of the present invention preferentially supports acetic acid-assimilating methane bacteria that consume volatile fatty acids and generate methane gas. Therefore, methane gas can be generated while volatile fatty acids are consumed and volatile fatty acids are prevented from accumulating in the methane fermentation liquid. Moreover, as a result of the consumption of volatile fatty acids, which are the decomposition products of cellulose, the reaction of generating volatile fatty acids from cellulose easily proceeds. Therefore, the progress of the decomposition reaction of cellulose can be promoted.

  In addition, when hydrogen-assimilating methane bacteria are supported on the microorganism-supporting carrier, methane gas can be generated from hydrogen and carbon dioxide, which are degradation products of cellulose. Further, as a result of the consumption of hydrogen and carbon dioxide, which are degradation products of cellulose, it becomes easier to proceed the reaction in which hydrogen and carbon dioxide are produced from cellulose. Therefore, the progress of the decomposition reaction of cellulose can be promoted.

  Furthermore, by supporting the saccharide-decomposing bacteria on the microorganism-supporting carrier, it is easy to promote the reaction in which saccharides are produced from cellulose as a result of degradation and consumption of saccharides such as glucose, which are degradation products of cellulose. . Therefore, the progress of the decomposition reaction of cellulose can be promoted.

  In addition, since the microorganism-supported carrier prepared using methane fermentation sludge as a microbial community carries anaerobic cellulose-degrading bacteria, acetic acid-assimilating methane bacteria, hydrogen-assimilating methane bacteria, and saccharolytic bacteria, Methane gas production from lignocellulosic biomass can be carried out very efficiently. Moreover, in this case, various bacterial communities are supported on the microorganism-supporting carrier. It is considered that this diverse bacterial community contributes in some way to the improvement of methane gas production efficiency from lignocellulosic biomass.

  The microorganism-supported carrier obtained by the production method of the present invention may be added to water, preferably water in which inorganic salts and the like that are preferable for the growth of microorganisms are dissolved, and methane fermentation may be performed. You may make it add to the methane fermentation liquid of a methane fermenter and perform methane fermentation. Moreover, you may make it perform the preparation method of the microorganisms support | carrier of this invention, and the methane gas production | generation method continuously. That is, lignocellulosic biomass is added to advance methane fermentation while the microorganism-supported carrier obtained by the production method of the present invention is in contact (immersed) with the suspension to which the microorganism community is added. Good.

  The lignocellulosic biomass can be the same as described above. According to experiments by the inventors of the present application, as lignocellulosic biomass, rice straw containing 32 to 37% cellulose, 29 to 37% hemicellulose and 5 to 15% lignin by dry weight can be directly subjected to methane fermentation. Methane gas was generated efficiently. Therefore, although it is considered that the present invention can be applied to lignocellulosic biomass in general, lignocellulosic biomass such as straw having a composition similar to or similar to this composition can be particularly preferably used. In order to promote the cellulose decomposition reaction by increasing the contact area with the methane fermentation liquor, pulverization is preferable, but the methane fermentation may be performed untreated without pulverization.

  The above-described embodiment is an example of a preferred embodiment of the present invention, but is not limited thereto, and various modifications can be made without departing from the gist of the present invention. For example, in the above-described embodiment, lignocellulosic biomass is treated with methane fermentation using a hydrophobic carrier on which at least anaerobic cellulose-degrading bacteria and acetic acid-assimilating methane bacteria are supported. A hydrophobic carrier carrying only methane bacteria may be introduced into the methane fermentation solution, and lignocellulosic biomass may be introduced into the methane fermentation solution for methane fermentation treatment. Since the methane fermentation broth contains anaerobic cellulose-degrading bacteria, by introducing a hydrophobic carrier carrying acetic acid-assimilating methane bacteria into the methane fermentation liquid, while decomposing lignocellulosic biomass, It is possible to efficiently generate methane gas while suppressing the accumulation of volatile fatty acids. Moreover, during the methane fermentation treatment, the anaerobic cellulose-degrading bacteria in the methane fermentation solution adhere to the carrier, and further, the bacterial community carried on the carrier is diversified. The methane gas can be generated efficiently.

  In addition, the hydrophobic carrier on which only acetic acid-assimilating methane bacteria are supported is, for example, acetic acid-assimilating methane in a suspension to which acetic acid-assimilating methane bacteria or a microbial community containing acetic acid-assimilating methane bacteria is added. It can be produced by adding a volatile fatty acid which is a substance that becomes a methane production source of the fungus, and immersing a hydrophobic carrier capable of supporting the same microorganism as described above in a suspension. The contact time between the suspension and the hydrophobic carrier can be determined by the same method as described above. Moreover, the same thing as the above can be used also about lignocellulosic biomass.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

<Example 1>
In the reactor, rice straw substrate, methane fermentation broth and carbon fiber nonwoven fabric were housed, and the production of methane gas from rice straw was verified.

<Comparative Example 1>
Except that no carbon fiber nonwoven fabric was housed in the reactor, methane gas production from rice straw was verified under the same conditions as in Example 1.

<Experiment method>
Example 1 and Comparative Example 1 were carried out by the methods specifically described below.

(1) Preparation of rice straw substrate Rice straw obtained from a Japanese farm was shredded to less than 2 mm. A rice straw substrate was prepared by blending this rice straw as follows. The yeast extract manufactured by Wako Pure Chemical Industries, Ltd. is used, and DSMZ Medium 131 trace element solution (hereinafter referred to as trace element solution) and DSMZ Medium 141 vitamin solution (hereinafter referred to as vitamin solution) are DSMZ ( Deutsche Sammlung von Mikroorganismen and Zellkulturen) was used.
[Composition of rice straw substrate (composition in 1 liter of water)]
Inaba: 10 g / L
KH ₂ PO ₄ : 0.8 g / L
K ₂ HPO ₄ : 1.6 g / L
NH ₄ Cl: 1 g / L
NaHCO ₃ : 2 g / L
MgCl ₂ · 6H ₂ O: 0.1 g / L
CaCl ₂ · 2H ₂ O: 0.2 g / L
NaCl: 0.8 g / L
Yeast extract: 1 g / L
Trace element solution: 10 mL / L
Vitamin solution: 10mL / L

  The rice straw substrate CODcr (chemical oxygen demand with dichromate) was approximately 11870 mg CODcr / L.

(2) Configuration of reactor A 250 mL glass bottle manufactured by Duran was used as the reactor. Six glass bottles were prepared, three of which were used in the experiment of Example 1 and the remaining three were used in the experiment of Comparative Example 1. In each of these glass bottles, an equal amount (200 g) of each sludge collected from the thermophilic anaerobic digester (methane fermentation tank) where stable gas generation from raw garbage was performed, and each rice bran substrate was added. 50 mL was added.

  In the three glass bottles used in the experiment of Example 1, two more carbon fiber nonwoven fabrics (type: pitch, porosity: about 98%, diameter: 30.0 mm, height: 70.0 mm, thickness: 2.4 mm, hereinafter referred to as CFT).

  Each glass bottle contained a stirrer for operating the reactor while stirring.

  Each glass bottle was filled with all the contents and then replaced with nitrogen gas and sealed with a silicone rubber stopper to ensure an anaerobic environment in the glass bottle.

(3) Reactor operating conditions The reactor was operated in semi-batch mode. Specifically, from the start of the experiment to the 15th day, 50 mL of the contents in the glass bottle were extracted once every 3 days using a syringe, and 50 mL of rice straw substrate was newly added. This operation was performed once every two days from the 16th day to the 45th day. Under these operating conditions, the OLR (organic load, ♦ in FIG. 1) and HRT (hydraulic residence time, ♦ in FIG. 1) during the experiment were as shown in FIG.

  Moreover, when performing the said operation, 0.5N NaOH aqueous solution was added suitably and the pH of the content in a glass bottle was maintained at 7.5 vicinity. The reactor was maintained at 55 ° C. during the experimental period. During the experiment period, the contents in the glass bottle were continuously stirred with an electromagnetic stirrer.

(4) Chemical component analysis The amount of biogas produced was measured by the water displacement method. Biogas composition analysis was performed by gas chromatography (Agilent, apparatus name: 6890N).

  Moreover, the lower fatty acid concentration and glucose concentration were analyzed about the contents in the glass bottle extracted with the syringe. The volatile fatty acid concentration analysis was performed by liquid chromatography (manufactured by GL Sciences, apparatus name: GL-7400). The glucose concentration analysis was performed using a spectrophotometer by the anthrone method.

  Further, COD (chemical oxygen demand) and SS (floating solid content) were analyzed for the contents in the glass bottle after the end of the experiment (day 45). The COD analysis was performed according to JIS K 0120-20 (HACH, device name: DR800). The SS analysis was performed according to JIS K0102-14.1 (manufactured by Yamato, device name DN63).

(5) Biological analysis After the end of the experiment (day 45), the contents were collected from each glass bottle and subjected to biological analysis. In addition, CFT was immersed in phosphate buffered saline and shaken, and the CFT deposit was transferred to phosphate buffered saline for biological analysis. The amount of 16S rRNA gene copy number of the microorganism group contained in these samples was quantitatively analyzed using real-time PCR. Specifically, the collected sample was centrifuged to settle the microorganism group, and suspended in Tris-EDTA buffer (pH 8.0, 100 mM Tris HCl, 40 mM EDTA). DNA was extracted by repeated bead collisions from the microorganism group in the presence of sodium dodecyl sulfate and a phenol-chloroform-isoamyl alcohol solution (25: 24: 1 v / v), and then the extracted DNA was extracted from the QIAamp DNA micro kit (Qiagen). Purified). This was subjected to TaqMan real-time PCT to quantitatively analyze the copy number of 16S rRNA gene of the microorganism group. The primer / probe set was as follows. This primer / probe set is described in the following paper (Takai, K., K. Horikoshi (2000). "Rapid detection and quatification of members of archaeal community by quantitative PCR using fluorogenic probes. "Appl. Environ. Microbiol. 66 (11): 5066-5072., Sawayama, S., Tsukahara K, Yagishita T (2006)." Phylogenetic description of immobilized methanogenic community using real-time PCR in a fixed-bed anaerobic digester . "Bioresour. Technol. 97 (1): 69-76.).
Prokaryotic primer / probe set:
Uni340F / Uni806R / Uni516F
・ Primer / probe set for methane bacteria:
SP-MArch-0348-Sa-17 / SD-Arch-0786-Aa-20 / SP-MArch-0515-Sa-25

  Further, by analyzing the terminal fragment length polymorphism (T-RFLP), the community structure of bacteria excluding archaea out of all bacteria and the community structure of archaea excluding bacteria among all bacteria were analyzed. Specifically, Ba27f labeled with 6-FAM at the 5 'end is used as a bacterial forward primer, Ba907r is used as a reverse primer, Ar109f is used as an archaeal forward primer, and a 5' end is used as a reverse primer. PCR was performed in a 50 μL reaction mixture using Ar912rt labeled with 6-FAM in to obtain a PCR amplicon. The PCR amplicon was purified by Wizard SV Gel and PCR Clean-Up System (Promega), MspI (New England BioLabs) was used as a bacterial restriction enzyme, and TaqI (New England BioLabs) was used as an archaeal restriction enzyme. England BioLabs). A DNA size standard GeneScan-500 ROX Size Standard (Applied Biosystems) was used with the purified digest as an internal size standard. Terminal fragment length polymorphism analysis was performed using 3130xl Genetic Analyzer (Applied Biosystems). Ingredients below 2% were removed from the data.

  In addition, clonal analysis was performed. Specifically, PCR was performed under the same conditions as for T-RFLP, and the PCR product was purified and ligated into a pGEM-T Easy vector (Promega). The plasmid was incorporated into Escherichia coli JM109 cells, and clones were randomly selected and sequenced by ABI 3130xl sequencer (Applied Biosystems) using BigDye Terminator cycle sequencing chemistry.

<Experimental result>
(1) Gas component analysis results FIG. 2 shows the results of Example 1 and Comparative Example 1 with respect to the change over time in the gas generation rate. From the start of the experiment (day 0) to the 15th day, the gas generation rate in Example 1 was slightly faster, but no significant difference was observed. However, after the 16th day when the load was increased by setting the HRT to 10 days, a significant difference was observed in the gas generation rate. That is, in Comparative Example 1, although the gas generation rate gradually decreased and decreased to 73 mL / L / day on the final day (45th day), in Example 1, the gas generation rate was increased to the final day. It gradually increased and finally increased to 257 mL / L / day.

  Furthermore, when the methane concentration in the gas on the last day (the 45th day) was measured, in Comparative Example 1, the methane concentration (volume) was 56 ± 1%, whereas in Example 1, the methane concentration (volume). Was 72 ± 2%, and it was revealed that Example 1 has an overwhelmingly higher methane concentration.

  From the above results, by introducing CFT into the reactor containing the methane fermentation broth, the effect of improving the gas production rate from rice straw and the effect of enabling operation at a higher load (at least HRT 10 days) It has become clear that there is an effect of improving the methane concentration in the gas (an effect of improving the amount of methane production).

(2) Results of volatile fatty acid concentration analysis The results of Example 1 and Comparative Example 1 are shown in FIG. 3 with respect to changes in the volatile fatty acid (acetic acid, propionic acid, butyric acid) concentration over time. In Example 1, almost no volatile fatty acid was detected over the entire period. On the other hand, in Comparative Example 1, volatile fatty acids were detected during the entire period, and in particular, there was a tendency for the concentration to increase as the experiment progressed to the second half. In addition, it was confirmed that most of the volatile fatty acid accumulated in Comparative Example 1 was acetic acid.

  From the above results, it is clear that by introducing CFT into the reactor containing the methane fermentation broth, it is possible to obtain the effect of preventing the accumulation of volatile fatty acids in the methane fermentation broth that is the contents in the reactor. It was.

  In addition, taking the above gas component analysis results into consideration, in Comparative Example 1, the decomposition of cellulose was inhibited by the accumulation of volatile fatty acids, and as a result, the gas generation rate became very low. Since there was almost no accumulation of volatile fatty acids, the decomposition of cellulose was rather accelerated without being inhibited, and as a result, the gas generation rate was considered to be improved.

(3) COD Analysis and SS Analysis Results Regarding the COD removal efficiency and SS removal efficiency, the results of Example 1 and Comparative Example 1 are shown in FIGS. 4 and 5, respectively. As apparent from FIGS. 4 and 5, Example 1 was overwhelmingly superior in COD removal efficiency and SS removal efficiency.

  From the above results, it became clear that by introducing CFT into the reactor containing the methane fermentation broth, the effect of improving the COD removal efficiency and the SS removal efficiency, that is, the effect of improving the rice straw decomposition efficiency was achieved. .

(4) Glucose concentration analysis results FIG. 6 shows the results of Example 1 and Comparative Example 1 regarding the glucose concentration. There was little difference in glucose concentration during the experiment.

  However, as described above, in consideration of the effect of improving rice straw decomposition efficiency by introducing CFT into the reactor containing the methane fermentation broth, in practice, Example 1 has more glucose. It was thought that it was disassembling. That is, if the decomposition efficiency of rice straw is improved, the production amount of glucose, which is a decomposition product of rice straw, is also increased. Therefore, it is considered that Example 1 had a larger amount of glucose production than Comparative Example 1. Nevertheless, during the experimental period, the fact that there was no difference in the glucose concentration between the two was the same as in Comparative Example 1 in which the amount of glucose produced was smaller in Example 1 due to more decomposition of glucose. It was thought that it was able to reduce to the glucose concentration of.

  From the above results, it was clarified that by introducing CFT into the reactor containing the methane fermentation broth, it was possible to improve the resolution of glucose, which is a decomposition product of rice straw.

(5) Quantitative PCR results of prokaryotes and methane bacteria The copy number of the prokaryotes and methane bacteria present in the fermentation liquid of Comparative Example 1 and the fermentation liquid of Example 1 by quantitative PCR, retained in the CFT of Example 1 Table 1 shows the results of confirming the copy number of the prokaryote and methane bacteria by quantitative PCR.

  There was no significant difference in the copy number by quantitative PCR of prokaryotes and methane bacteria present in the fermented liquid of Comparative Example 1 and the fermented liquid of Example 1. From this, it was suggested that the difference in the experimental results between Example 1 and Comparative Example 1 was achieved by the microorganisms supported on CFT.

(6) Comparison of archaeal community structure by T-RFLP By end fragment length polymorphism analysis (T-RFLP), the archaea community structure excluding bacteria was compared among all bacteria. The results are shown in FIG. In FIG. 7, A is the result regarding the bacteria in the fermented liquid of Comparative Example 1, B is the result of the bacteria in the fermented liquid of Example 1, and C is the result regarding the bacteria retained on the CFT of Example 1.

  Table 2 shows the results of analyzing the structure of archaeal communities by TA cloning.

  From the results shown in FIG. 7 and Table 2, Methanosarcina thermophila belonging to the genus Methanosarcina is dominantly retained on the CFT, and further, Methanothermobacter thermautotrophicus belonging to the genus Methanothermobacter. Methanobacterium formicicum belonging to the genus of bacteria (Methanobacterium) was retained. In particular, Methanosarcina thermophila belonging to the genus Methanosarcina was retained on CFT at an overwhelmingly high rate compared to the fermentation broth.

  Here, the microorganism belonging to the genus Methanosarcina is an acetic acid-assimilating methane bacterium, and the microorganisms belonging to the genus Methanotherbacter and the genus Methanobacteria are hydrogen-assimilating methane bacteria. Therefore, it has been clarified that acetic acid-assimilating methane bacteria can be preferentially supported on CFT, and further, hydrogen-assimilating methane bacteria can also be supported.

  And as above-mentioned, in Example 1, it was thought that accumulation | storage of a volatile fatty acid was suppressed and methane gas production | generation continued. Considering this, as a result of bringing CFT into contact with the fermentation broth, it is possible to preferentially carry acetic acid-assimilating methane bacteria on the CFT, and as a result, accumulation of volatile fatty acids in the fermentation broth is suppressed. It was thought that methane gas production could be sustained.

  Furthermore, it was clarified from this result that acetic acid-assimilating methane bacteria are easier to maintain and grow in a hydrophobic environment than in a hydrophilic environment such as in a fermentation broth. Therefore, it was considered that not only CFT but also acetic acid-assimilating methane bacteria can be preferentially supported on a hydrophobic carrier such as polyethylene or polypropylene. It was also considered that hydrogen-utilizing methane bacteria could be retained on a hydrophobic carrier.

(7) Comparison of bacterial community structure by T-RFLP By terminal fragment length polymorphism analysis (T-RFLP), the bacterial community structure of all bacteria except archaea was compared. The results are shown in FIG. In FIG. 8, A is the result regarding the bacteria in the fermented liquid of Comparative Example 1, B is the result of the bacteria in the fermented liquid of Example 1, and C is the result regarding the bacteria retained on the CFT of Example 1.

  Table 3 shows the results of analyzing the structure of the bacterial community by TA cloning.

  First, from the results of TA cloning, Clostridium thermocellum related species (85.86% homology) and Clostridium cellulolyticum related species (81% homology) are retained on CFT. It had been. Moreover, Anaerobaculum mobile which is a microorganism belonging to the genus Anaerobaculum was retained.

  Here, microorganisms belonging to the genus Clostridium are anaerobic cellulose-degrading bacteria. A microorganism belonging to the genus Anaerobaculum is a saccharide-decomposing bacterium that degrades saccharides, particularly glucose. Therefore, it has been clarified that an anaerobic cellulose-decomposing bacterium can be supported on the CFT, and further, a saccharide-decomposing bacterium can be supported.

  Further, from the results shown in FIG. 8, there were 8 fragments detected in A, whereas 10 fragments in B and 11 fragments in C. The total number of fragments obtained in B and C was 14 It was confirmed to be a seed. From this result, it was clarified that bacteria were diversified in the reactor equipped with CFT.

  In any of A, B, and C, six types of fragments of 84, 92, 148, 262, 464, and 545 bp were detected. In contrast, in B and C, 8 types of fragments of 82, 91, 132, 199, 270, 277, 280 and 465 bp that were not detected in A were detected. Therefore, it was revealed that the bacterial community of Example 1 with CFT was clearly different from the bacterial community of Comparative Example 1 without CFT.

  Therefore, it was suggested that by using methane fermentation sludge as the microbial community and adding CFT to the methane fermentation solution, the bacterial community can be diversified on CFT to promote cellulose degradation.

Claims (10)

Adding a carrier capable of supporting microorganisms to a suspension to which a microorganism community including at least anaerobic cellulose-degrading bacteria and acetic acid-assimilating methane bacteria is added, and adding lignocellulosic biomass to the suspension; A method for producing a microorganism-supporting carrier for producing methane gas from lignocellulosic biomass, comprising a step of causing a decomposition reaction of the lignocellulosic biomass by the microbial community. The method for producing a microorganism-supporting carrier according to claim 1, wherein the microorganism community further contains at least one of hydrogen-utilizing methane bacteria and saccharolytic bacteria. The method for producing a microorganism-supporting carrier according to claim 3, wherein the suspension is methane fermentation sludge or a methane fermentation sludge-containing liquid. The method for producing a microorganism-supporting carrier according to any one of claims 1 to 3, wherein the hydrophobic carrier is a carbon carrier. The method for producing a microorganism-supporting carrier according to any one of claims 1 to 4, wherein the lignocellulosic biomass is straw. The method for producing a microorganism-supporting carrier according to claim 5, wherein the lignocellulosic biomass is rice straw. A method for producing methane gas from lignocellulosic biomass, comprising subjecting lignocellulosic biomass to methane fermentation using the microorganism-supported carrier obtained by the method according to any one of claims 1 to 6. A method for producing methane gas from lignocellulosic biomass, comprising charging a methane fermentation broth with a hydrophobic carrier on which acetic acid-assimilating methane bacteria are supported and lignocellulosic biomass. The method for producing methane gas according to claim 7 or 8, wherein the lignocellulosic biomass is straw. The method for producing methane gas according to claim 9, wherein the lignocellulosic biomass is rice straw.

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